Project Summary Kidney podocytes operate under substantial biomechanical stress as part of their physiological function to filter plasma. They have a specialized dynamic actin cytoskeleton structure to reinforce the fragile foot process morphology that is required for their function. Using in vivo proteomic screening and mining of NephroSeq data, we have identified nebulette as a novel scaffolding protein that may play a role in organization and maintenance of the podocyte cytoskeleton and adhesion. Nebulette is an actin-binding protein expressed highly in the heart; mutations on its nebulin repeat domains have been linked to abnormal cardiac biomechanics and dilated cardiac hypertrophy. We hypothesize that nebulette plays a critical role in stability of actin filaments in kidney podocytes, increasing their biomechanical resilience against injury. We will test this hypothesis in vitro and in vivo using cultured primary podocytes and podocyte-specific nebulette knockout mice, respectively. We will use high-content imaging to characterize morphological changes associated with nebulette ablation and live-cell microscopy to determine its effects on cell motility and calcium dynamics. We will also quantitatively characterize cytoskeletal biomechanics using atomic force microscope elastography. We will then use proteomics and network analyses to construct a spatially specific partial differential equations- based dynamical model to quantify contribution of different biphysical components of nebulette in cytoskeletal stability and focal adhesion distribution and function. This in silico process will also identify likely drug targets that may modulate podocyte mechanobiology in a cell-specific manner. We will finally test the contribution of nebulette to in vivo podocyte physiology using two different disease models in podocyte-specific nebulette knockout mice. Through these experiments and computational modeling, we will thoroughly characterize the role of nebulette in glomerular physiology, and potentially identify novel pathways to modify biomechanical resilience of podocytes under healthy and disease conditions.